N-(2-Bromo-4-fluorophenyl)-3-(3,4-dihydroxyphenyl)-acrylamide (CPAM), a small catecholic amide as an antioxidant, anti diabetic and antibacterial compound

Abstract

The trans conformation of N-(2-bromo-4-fluorophenyl)-3-(3,4-dihydroxyphenyl)-acrylamide (CPAM), a small catecholic amide with halogen moieties, may be used as an anti diabetic compound. This newly synthesized CPAM was found to reduce blood sugar levels about 3 fold in streptozotocin (STZ) induced diabetic rats at a dose of 2.5 mg kg⁻¹ body weight after 15 days of treatment. A post diabetic study revealed that CPAM could normalize alkaline phosphatase (ALP), acid phosphatase (ACP) and prostatic ACP values. The ED₅₀ and ED₉₀ values of CPAM against rats were determined as 1 mg kg⁻¹ and 2.5 mg kg⁻¹ body weight respectively. No adverse toxicological effects of CPAM were noted on the body weight, blood, liver or kidneys of the experimental animals. Furthermore, it was observed from histopathological results that CPAM had no unfavourable effects on the vital organs of the rats. The presence of CPAM in the blood serum of tested rats also ensured its wide range of applicability. In addition, the synthetic CPAM compound showed antibacterial activity against Mycobacterium smegmatis, Pseudomonas aeruginosa, Staphylococcus aureus and clinically drug resistant S. aureus (MRSA) strains. Thus, CPAM might be useful for the treatment of secondary infections which are common for diabetic individuals.

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1. Introduction

Catechol and its derivatives are well known as antioxidants. They promote dehydrogenative one electron oxidation processes, and can behave as electrophilic or nucleophilic entities in biological systems so helping to reduce the risk of several degenerative diseases by absorbing undesirable free radicals.^1–7 Density functional theory calculations reveal that the presence of at least two adjacent hydroxy groups on a phenyl ring containing an electron withdrawing moiety (e.g. in caffeic acid) have relatively weak bond dissociation energies for the phenolic-OH bonds. This enables the production of phenoxy radicals, which are stabilized by the delocalization of the unpaired electron through extended conjugation, and act as potent antioxidants.^8,9

Diabetes mellitus (DM) is a chronic disease associated with carbohydrate metabolism and is caused by a deficiency in insulin secretion or by ineffectiveness in insulin action. The International Diabetes Federation has estimated that the total number of diabetic patients could be about 439 million by 2030.¹⁰ For decades, several research groups working on anti diabetic agents have reported that cinnamic acid and substituted cinnamic acids (e.g. caffeic acid and isoferulic acid) exhibit antihyperglycemic activities; they enhance blood glucose uptake by activation of α1-adrenoceptors, and increase glucose uptake in normal FL83B cells.^11,12 Liao et al. suggested that caffeic acid could increase β-oxidation by activation of 5′ AMP-activated protein kinase (AMPK) in liver and also could improve high fat diet (HFD) induced obesity in mice.¹³

An amide bond is a common linkage in commercially available anti diabetic drugs such as sulfonylurea and thiazolidinedione. Sulfonylurea groups increase insulin receptor sensitivity and delay digestion, whereas thiazolidinedione groups act by activating peroxisome proliferator-activated receptors.^14,15 However, these agents can produce severe hypoglycemia, weight gain, and gastrointestinal disturbances.¹⁶ Tabatabaie et al. reported that over-expression of cyclooxygenase-2 (COX-2) forms reactive oxygen species, which are known to damage β-cells.¹⁷ Prolonged exposure to reactive oxygen species may be a contributing factor to the eventual reduction in manganese superoxide dismutase (MnSOD) gene expression and activity in diabetic rat aorta.¹⁸ Therefore, the treatment of chronic diseases resulting from oxidative stress requires the use of antioxidant agents to improve endothelial function.¹⁹ Amide derivatives of caffeic acid have many pharmacological activities such as antioxidant and anti-platelet effects, and inhibitory effects on prostaglandin (PG) synthetase, matrix metallopeptidase (MMP)-2, MMP-9 and arachidonate 5-lipoxygenase.^20,21

Additionally, diabetic patients are more susceptible to attack by various multidrug resistant bacteria that are posing a major concern in global health.^22–25 The current antibiotics are not very effective against multidrug resistant bacteria, which make some bacterial diseases untreatable.^26,27 This situation can be controlled by limiting the indiscriminate use of conventional antibiotics and employing proper therapeutic strategies with a new class of synthetic antimicrobial drugs. Currently, a few strains of Staphylococcus aureus (S. aureus) can be treated with vancomycin but for certain drug resistant strains of Mycobacterium tuberculosis and vancomycin-resistant enterococci, no viable alternatives exist.²⁸ In 2010, Zhu et al. reported that a series of caffeic acid amides had considerable antibacterial activities against Bacillus subtilis.²⁹

Based on all of these studies, the role of N-(2-bromo-4-fluorophenyl)-3-(3,4-dihydroxyphenyl)-acrylamide (CPAM) as an antioxidant, anti diabetic and antibacterial agent was investigated. The anti diabetic properties of CPAM were studied on adult male albino rats (Wistar strain), whereas antimicrobial studies were carried out against Mycobacterium smegmatis (M. smegmatis), Pseudomonas aeruginosa (P. aeruginosa), S. aureus, and drug resistant S. aureus strains including MRSA.

2. Materials and methods

2.1 Materials

Melting points were determined in open-ended capillary tubes. Caffeic acid (3,4-dihydroxycinnamic acid) and the amine 2-bromo-4-fluoroaniline were purchased from Sigma (India). Other fine chemicals were obtained from commercial suppliers and were used without further purification. Solvents were dried and distilled in accordance with standard procedures. TLC was carried out on precoated plates (Merck silica gel 60, F₂₅₄), and the spots were visualized with UV light or by charring the plates by dipping them in 5% sulphuric acid–methanol (H₂SO₄–MeOH), 2,4-dinitrophenylhydrazine (2,4-DNP) and ninhydrin solution. Column chromatography was performed on silica gel (230–400 mesh). ¹H and ¹³C NMR for CPAM were recorded at 600 MHz using deuterated dimethyl sulfoxide (DMSO-d₆) as the solvent. The multiplicity of the peaks are marked as s-singlet, d-doublet, t-triplet, q-quartet, m-multiplet and bs-broad singlet. High Resolution Mass Spectra (HRMS) were recorded using a quadrupole-equipped time-of-flight (ToF) mass spectrometer.

2.2 Synthesis of CPAM

To a well-stirred solution of caffeic acid (180 mg, 1 mmol) in dry tetrahydrofuran (THF, 10 ml), N,N′-dicyclohexylcarbodiimide (DCC, 226 mg, 1.1 mmol) and 2-bromo-4-fluorophenylamine (209 mg, 1.1 mmol) were added. The mixture was stirred at room temperature for 16 h under a nitrogen atmosphere. The reaction mixture was filtered and the filtrate was concentrated under vacuum. The concentrated gummy layer was then treated with a saturated solution of NaHCO₃ and the aqueous part was neutralized with dilute HCl. The aqueous layer was then extracted with ethyl acetate (3 × 100 ml). The organic phase was demoisturized with anhydrous sodium sulfate and evaporated under reduced pressure. After that, the reaction mixture was purified by flash chromatography using ethyl acetate and n-hexane as the eluent with an isolation yield of 71% (250 mg). The final product was recrystallized by ethanol.

2.3 Evaluation of radical scavenging properties by DPPH assay

The synthesized amide was tested for radical scavenging activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical.³⁰ DPPH in ethanol (100 μM, 2 ml) was added to 2 ml of test compounds in ethanol (caffeic acid and CPAM) and final concentrations of test compounds were adjusted from 16 μM to 4 μM by adding ethanol. Each mixture was shaken vigorously and kept for 30 min at room temperature in the dark. The decrease in absorbance of DPPH at 517 nm was measured. Ethanol was used as the blank solution. DPPH solution (2 ml) in ethanol (2 ml) served as the control. The radical scavenging activities of the samples were expressed in terms of EC₅₀ values (concentration in μM required for a 50% decreased in absorbance of the DPPH solution). The percentage inhibition of DPPH with various concentrations of test samples was calculated using eqn (1).

% Inhibition = [{(A_control − A_test)/A_control} × 100] (1)

where A_control is the absorbance of the control (DPPH without CPAM) and A_test is the absorbance of the test sample in DPPH solution. A plot of % inhibition vs. concentration was made to establish the standard curves and to calculate EC₅₀ values of the synthetic amide CPAM, and caffeic acid.

2.4 Toxicity analysis

Eighteen adult male albino rats (Wistar strain) having standard body weights (145–175 g) and ages (50–70 days) were procured from the animal housing facility ​(Jadavpur University). The animals were acclimatized under standard conditions of temperature and humidity with 12 h light dark cycles. They were housed in polypropylene cages and were fed with a normal protein diet (18% casein, 70% carbohydrate, 7% fat, 4% salt mixture and 1% vitamin mixture).³¹ The animals were divided into 6 groups of 3 rats. Group A was a control group; animals in this group were not treated with CPAM. Group B to Group F were considered as experimental groups in which animals were injected daily with CPAM at doses of 1 mg (Group B), 2 mg (Group C), 3 mg (Group D), 5 mg (Group E), and 10 mg (Group F) per kg body weight for 15 days. All the rats were given their respective doses of CPAM intraperitoneally in 0.5 ml of 60% ethanol–water and at the same time every day. The animals in Group A were given the same volume of 60% ethanol–water during this period. After administration of the CPAM, the gross activity, posture and tone, eye ball movement, reaction and reflexes as well as mortality of the animals were noted every day. On the 15th day, the rats were sacrificed and blood was collected from the heart for toxicological analysis. The total haemoglobin, serum acid phosphatase (ACP), serum alkaline phosphatase (ALP), serum glutamate pyruvate transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), urea, and creatinine levels of the CPAM treated animals were determined for toxicological analysis.

2.4.1 Biochemical analysis. Haemoglobin was measured using a Sahli Haemometer, Superior, W. Germany, by taking 20 μl blood and diluting it with 0.1 N HCl until the colour was matched with the given standard (reference value is 14 to 16 g dl⁻¹). ACP was measured by using the kit provided by Accurex Biomedical Pvt. Ltd., Thane, India (reference value is up to 8 IU l⁻¹ at 37 °C). ALP, SGPT, SGOT and urea were measured using individual kits provided by Piramal Healthcare Limited, Mumbai, India (reference values of these parameters are up to 12 IU l⁻¹, 40–60 U l⁻¹, 150–200 U l⁻¹ and <40 mg dl⁻¹ respectively at 37 °C). Creatinine was determined from a kit provided by Merckotest®, Merck, Goa, India, using the modified Jaffe’s method (reference value is 0.5–1.1 mg dl⁻¹).

2.5 Experimental design for assessment of diabetic rats

Male albino rats (fasting blood sugar levels 80–120 mg dl⁻¹) were procured from the animal housing facility. The animals were intraperitonially injected with streptozotocin (STZ) (60 mg kg⁻¹ body weight, dissolved in 0.1 M citrate buffer at pH 4.5) to induce diabetes.³² The induced diabetes was confirmed by measuring the fasting blood sugar level (>200 mg dl⁻¹) after 3 days. A total of 56 diabetes induced animals were clustered into 7 groups of 8 rats, designated Groups 2–8. Group 1, control group in which animals were not treated with either STZ or CPAM. Group 2, was the diabetic control group. Group 3, was a positive control group, i.e. rats were treated with the diabetic drug metformin hydrochloride (500 mg kg⁻¹ body weight) for 15 days. Group 4 to Group 8 were experimental diabetic groups in which animals were treated with CPAM at different daily doses (0.5, 1.0, 1.5, 2.0 and 2.5 mg kg⁻¹ body weight, respectively) for 15 days. All the rats were given their respective doses of CPAM intraperitoneally in 0.5 ml of 60% ethanol–water at 8 am every day. The blood sugar level in 12 h fasting condition of each rat was measured after 5 days interval till the completion of the experiment. Body weight and general health conditions were also monitored every day in the fasting state.

2.5.1 Blood glucose assay. The blood glucose level of each rat was measured by making a small incision at the tip of the tail. The blood glucose was measured in 12 h fasting conditions using a digital Breeze 2 glucometer supplied by Bayer Healthcare LLC, USA (a normal fasting blood sugar reference value is <140 mg dl⁻¹).

2.6 Post diabetes liver parameter analysis

After completion of the diabetes experiment, the surviving rats were sacrificed on the 16th day (except for control group 1 and the metformin hydrochloride group 3) after mild anesthesia, and blood samples were collected from the hearts of the experimental rats treated with CPAM during the 15 day experiment. Serum samples were separated from the blood and stored at −60 °C in freeze (Jadavpur University). Blood samples were stored in normal BD Vacutainer and EDTA (5.4 mM/3 ml blood) containing BD Vacutainer for further analysis. ALP, ACP and prostatic ACP levels were determined for post diabetes liver parameter analysis.

2.6.1 Biochemical analysis of post diabetes rats. ALP determination was done using the kit provided by InnolineTM, Merck, Mumbai, India. The absorbance was taken at a wavelength of 405 nm against a baseline of distilled water (reference values of ALP using this kit are: men < 270 U l⁻¹, women < 240 U l⁻¹, rats < 280 U l⁻¹ at 37 °C). Total ACP and prostatic ACP were determined using the kit provided by Crest Biosystems, Goa, India. The absorbance readings were taken at 37 °C against distilled water (reference values of ACP using this kit are: men < 4.7 U l⁻¹, women < 3.7 U l⁻¹ and the reference value for prostatic ACP is <1.6 U l⁻¹ at 37 °C).

2.7 SOD assay

The antioxidant enzyme level was studied from the serum of the rats by assaying super oxide dismutase (SOD). SOD activity was determined by using the method described by Beauchamp and Fridovich in 1971 based on the reduction of nitroblue tetrazolium (NBT) to blue pharmazone by superoxides.³³

2.8 Histopathological experiment

Group 9 comprised 8 adult male albino rats (Wistar strain) separately used for histopathological experiments. The animals were divided into 4 groups of 2 rats. Group A, was an untreated control group and Group B was the diabetic control group (STZ induced). Group C and Group D were STZ induced diabetic rats where the animals were treated with 2.5 mg kg⁻¹ body weight CPAM or 500 mg kg⁻¹ body weight metformin hydrochloride, respectively, for 15 days. The drug and CPAM were intraperitoneally administered at 8 AM every day. At the end of this experiment, the rats were sacrificed and the organs (liver, pancreas and kidney) were collected and kept in saline containing 10% formalin. The organs were then embedded in paraffin, sectioned at 5 microns and stained with haematoxylin and eosin. The sections were examined for structural changes in the cells under bright field light microscopy (Axio Scope, A1, Zeiss, Germany).

2.9 Detection of CPAM in blood samples of experimental rats

Fifteen experimental rats were taken and clustered into 5 groups, having 3 rats in each group. Group A was the control group in which animals were not treated with CPAM. Animals in Group B to Group E were treated with CPAM at doses of 10 mg kg⁻¹ body weight. The doses were intraperitoneally administered to all rats in Groups B–E only once. The serum was collected from individual rats at different time intervals such as 1 h, 6 h, 12 h, and 24 h post-treatment. The serum samples were then lyophilized. For matrix-assisted laser desorption/ionization (MALDI) ToF analysis, solid serum samples were re-dissolved in 60% ethanol–water and centrifuged. An aliquot (4 μl) of supernatant was mixed into 4 μl of HCCA matrix (TA30 solvent containing 30 : 70 [v/v] acetonitrile : 0.1% TFA in water). Finally, 1.0 μl of each sample mixture was spotted onto the MALDI sample plate, and allowed to air-dry prior to the MALDI ToF analysis. The molecular masses of all these spots were determined using an Ultraflex ToF/ToF mass spectrometer equipped with a smart laser (Bruker, USA). The mass analysis was performed in positive, reflector ionization mode with a low mass scanning range of 200–600 m/z, and reproducibility was checked five times using separately spotted samples. For ¹H-NMR analysis, lyophilized crude serum samples were dissolved in DMSO-d₆ and ¹H-NMR spectra were recorded at 600 MHz (Bruker, USA).

2.10 CPAM antibacterial assay

The antimicrobial activities of CPAM were tested against S. aureus (ATCC-25923), P. aeruginosa (ATCC-15692/PAO1), MRSA (ATCC-43300) and M. smegmatis (ATCC-700084/mc2 155) by colony forming unit (CFU) assays. Exponentially grown bacterial cultures were centrifuged at 2300 g for 5 min and the pellets were suspended in LB or 7H9 media. Finally, the optical densities (ODs) of the samples were adjusted to 0.1 at 600 nm. Various concentrations of CPAM were incubated with equal amounts of bacteria in LB or 7H9 media in 96-well round bottom plates in triplicate. After the indicated time points, bacteria were harvested and the number of CFUs were assayed by plating serial dilutions on LB agar plates as described previously.³⁴ Bacterial colonies were enumerated after 24 h for S. aureus and P. aeruginosa and 72 h for M. smegmatis. All samples were plated in triplicate and values for three independent experiments were averaged.

2.11 Statistical analysis

Statistical analyses were performed by unpaired t test with GraphPad Prism v 5.0 [GraphPad Software, La Jolla, California USA, (http://www.graphpad.com/quickcalcs/ttest1.cfm?Format=SD)]. The two-tailed P value difference between groups was considered to be significant if P < 0.0001 (***), P < 0.001 (**) or P < 0.05 (*).

3. Results

3.1 Structural conformation of CPAM

CPAM was synthesized by a coupling method, in which caffeic acid and 2-bromo-4-fluorophenylamine were the starting materials and DCC was added as the coupling reagent in dry THF (Fig. 1A). The structure of CPAM was confirmed by spectroscopic as well as single crystal X-ray diffraction techniques. The crystal structure of CPAM showed that it was the trans conformer (Fig. 1B).

Fig. 1 (A) Synthesis of CPAM. (B) ORTEP view of CPAM.

3.2 CPAM as an antioxidant

The synthesized CPAM was tested for its radical scavenging activity using the DPPH radical. A plot of % inhibition vs. concentration was drawn to establish the standard curve and to calculate the EC₅₀ values of the synthetic amide, CPAM, and caffeic acid. The EC₅₀ values of CPAM and caffeic acid were determined as 10.25 ± 0.89 μM and 14.26 ± 1.07 μM, respectively.

3.3 Toxicological analysis of CPAM

For the toxicological analysis of CPAM, adult male albino rats (Wistar strain) were used, and CPAM was found to be safe: no mortality was observed among the animals treated with different doses of the compound (1 to 10 mg kg⁻¹ body weight) for 15 days; no animals showed visually apparent degradation of health; and the overall body weights of the animals remained constant during the course of treatment (Fig. 2A).

Fig. 2 Toxicological analysis of doses up to 10 mg kg⁻¹ body weight of CPAM administered daily to experimental rats for 15 days. (A) Body weight (g). (B) Haemoglobin (g dl⁻¹). (C) Serum acid phosphatase (IU l⁻¹). (D) Serum alkaline phosphatase (IU l⁻¹). (E) Serum glutamate pyruvate transaminase (U l⁻¹). (F) Serum glutamic oxaloacetic transaminase (U l⁻¹). (G) Urea (mg dl⁻¹). (H) Creatinine (mg dl⁻¹). Values are the means ± S.D. of three independent experiments.

There was no significant adverse effect of CPAM on blood haemoglobin as shown (Fig. 2B). ACP and ALP are the two important marker enzymes for the determination of liver injury; ACP levels in the experimental animals remained almost unaltered with respect to the control group (Fig. 2C) while ALP levels were observed to be slightly higher in the experimental animals than in the control group, particularly with higher doses, but all the values fell in the well-accepted range as per the supplier's reference (Fig. 2D). SGPT and SGOT levels in CPAM treated animals were increased with respect to the control animals but all these values were again found to be within the reference range (Fig. 2E and F).

The urea levels of the experimental rats initially decreased at lower doses of CPAM but then gradually increased with increasing doses of the compound with respect to the control group (Fig. 2G). An increasing trend for creatinine levels was observed from the beginning in the experimental animals with increasing doses of CPAM (Fig. 2H). However, the increased values for urea and creatinine also remained within the reference ranges.

3.4 Effect of CPAM on STZ induced diabetic rats

STZ induced diabetic animals were observed to have fasting blood sugar levels of more than 200 mg dl⁻¹ which decreased with CPAM treatment in a dose dependent manner. It was noted that only 25% animals survived in the untreated diabetic control group (Group 2) during the 15 day experiment (Table 1).

Table 1 Measurement of fasting blood sugar levels and survival percentages of diabetic rats with and without treatment^a

Groups	Day 0	Day 5	Day 10	Day 15	No. of rats surviving after 15 days	% of survival
a Each group contained 8 rats; data are means of 8 replicates ± S.D. The result obtained for metformin hydrochloride treatment (Group 3) was found to be statistically significant with p ≤ 0.001 (***) and those for CPAM were found to be statistically significant with p ≤ 0.05 (*), p ≤ 0.001 (**) or p ≤ 0.0001 (***), depending on dosage, compared to that of the diabetic control group (Group 2) without treatment.
Gr.1 control rats	98.56 ± 15.25	112.0 ± 22.21	108.8 ± 29.10	116.02 ± 32.29	7	87.5
Gr.2 diabetic control (STZ 60 mg kg^−1 body weight)	314.62 ± 59.23	330 ± 47.81	351 ± 51.26	386.50 ± 48.79	2	25
Gr.3 positive control (metformin hydrochloride 500 mg kg^−1 body weight)	332.25 ± 51.85	266.42 ± 51.69*	168.0 ± 50.80**	112.66 ± 27.86***	6	75
Gr.4 CPAM 0.5 mg kg^−1 body weight	305.75 ± 66.82	283 ± 40.64	263.66 ± 45.05	245.33 ± 52.48*	3	37.5
Gr.5 CPAM 1 mg kg^−1 body weight	334.12 ± 58.91	309.83 ± 69.51	218.25 ± 62.02*	209.25 ± 55.40*	4	50
Gr.6 CPAM 1.5 mg kg^−1 body weight	318.75 ± 69.72	210.66 ± 88.61	212.8 ± 65.44*	145.6 ± 62.67*	5	62.5
Gr.7 CPAM 2 mg kg^−1 body weight	322.12 ± 57.53	196.42 ± 74.40*	143.14 ± 48.86**	136.42 ± 47.90**	7	87.5
Gr.8 CPAM 2.5 mg kg^−1 body weight	307.25 ± 63.93	186.71 ± 72.70*	142.71 ± 46.20**	108.71 ± 21.01***	7	87.5

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Treatment with CPAM at a dose of 0.5 mg kg⁻¹ body weight had a low effect in decreasing the blood sugar of the experimental rats (Table 1). With an increase of the CPAM dose to 1 mg kg⁻¹ body weight, the fasting blood sugar level decreased from 334.12 ± 58.91 mg dl⁻¹ to 209.25 ± 55.40 mg dl⁻¹ (p ≤ 0.05) after the 15 day treatment period. It was observed that in Gr.6, 50% animals having blood sugar levels above 200 mg dl⁻¹ had those levels reduced to normal with 1.5 mg dose of CPAM over the length of the experiment (Table 1). Again, the fasting blood sugar levels of the experimental animals in Group 7 were continuously decreased by daily treatment with 2 mg kg⁻¹ body weight CPAM, and were reduced from 322.12 ± 57.53 mg dl⁻¹ to 136.42 ± 47.90 mg dl⁻¹ (p ≤ 0.001) after 15 days. Moreover, STZ induced diabetic rats having fasting blood sugar levels of 307.25 ± 63.93 mg dl⁻¹ at day zero had those levels reduced to 108.71 ± 21.01 mg dl⁻¹ (p ≤ 0.0001) after 15 days treatment with CPAM at a dose of 2.5 mg kg⁻¹ body weight. The effective dose 50 (ED₅₀) and effective dose 90 (ED₉₀) values were 1 mg kg⁻¹ body weight and 2.5 mg kg⁻¹ body weight respectively, determined from the data shown (Table 1). On increasing the dose from 0.5 mg to 2.5 mg, the survival percentage was increased from 37.5% to 87.5% (Table 1) and the decrease in fasting blood sugar went from 19% to 64% (ESI Table 1†). There was a 64.61% reduction in sugar levels at the dose of 2.5 mg CPAM. Post diabetes liver parameters were determined to investigate liver damage in the experimental rats after completion of the diabetic experiment. It was observed that treatment with CPAM not only reduced fasting blood sugar levels but also normalized the ALP, ACP, and prostatic ACP levels of the treated rats with respect to those of the diabetic control group (Group 2). As observed in Fig. 3A, ALP values were decreased with increasing CPAM dosage and became steady after a certain dosage. ACP and prostatic ACP levels of the treated rats were also reduced compared to the diabetic control values after treatment with CPAM, and these remained within the kit's normal reference range (Fig. 3B and C).

Fig. 3 Post diabetes liver parameter analysis after completion of the diabetes experiment (15 days). (A) Serum ALP (U l⁻¹). (B) Serum ACP (U l⁻¹). (C) Serum prostatic ACP (U l⁻¹). Values are the means ± S.D. The results obtained from the post diabetes experiment were found to be statistically significant (p ≤ 0.05 (*) and p ≤ 0.001 (**)) compared to those of the diabetic control group (Group 2) without treatment with CPAM.

3.5 Measurement of SOD in rat serum samples

With the induction of diabetes, SOD levels decreased as compared to those of the control group (Fig. 4).

Fig. 4 Measurement of SOD levels in diabetic animals and in treated groups after 15 days. Values are the means ± S.D. of three independent experiments.

However, in the presence of CPAM, the SOD levels were restored to some extent in the blood serum of the treated rats. A dose of 5 mg kg⁻¹ body weight of CPAM showed the highest restoration of SOD levels after 15 days of treatment (Fig. 4). The SOD levels of the group treated with 2.5 mg kg⁻¹ body weight of CPAM were comparatively higher with respect to the diabetic control group. The experimental results show that a standard dose of CPAM induces an antioxidant response in diabetic rats (Fig. 4).

3.6 Histopathological observations on different organs of experimental animals

Histopathological observations were made on the livers of control rats, diabetic rats, and rats treated with CPAM or the standard drug, metformin, for 15 days (Fig. 5A–D). Liver biopsies of the rats in the control group did not show any histological changes during the period of the experiment (15 days); the hepatocytes (liver cells) appeared normal with polygonal shapes (Fig. 5A).

Fig. 5 Histopathological pictures of rat liver cells after 15 days. (A) Control group. (B) Diabetic control group. (C) Group treated with 2.5 mg kg⁻¹ body weight of CPAM. (D) Group treated with 500 mg kg⁻¹ body weight of metformin hydrochloride. hepatocytes, cloudy swelling. 100×.

The morphology of the hepatocytes was abnormal and distorted in STZ induced diabetic rats with respect to those of the control group (Fig. 5B). Induced diabetic rats showed cloudy swellings in the hepatocytes (Fig. 5B). However, no abnormalities were seen in the hepatocytes of the experimental animals treated with CPAM (2.5 mg kg⁻¹ body weight) or metformin (500 mg kg⁻¹ body weight) after 15 days of treatment (Fig. 5C and D). The experimental results showed that CPAM had no adverse effect on the liver cells of the rats.

In pancreatic sections from the control rats, the acini were of regular shape showing the healthy state of the pancreatic cells after the 15 day treatment period. The islets of langerhans were normal with greater numbers of acini (Fig. 6A). However, in diabetic animals, the acini did not appear to be regular shaped in almost all cases; the cells were distorted and were also fewer in number with respect to control group (Fig. 6B). Cytoplasmic degenerative changes were also observed in the centres of the islets of langerhans and vascular congestion was observed near islets, suggesting an unhealthy pancreas. However, in the diabetic rats treated with CPAM at a dose of 2.5 mg kg⁻¹ body weight or with metformin, the acini and the islets of langerhans regained their healthy morphology (Fig. 6C and D). In addition, specimens from the CPAM treated group showed significantly more signs of recovery in the pancreatic cells suggesting that CPAM could have pancreatic cell revival traits with respect to diabetic control group.

Fig. 6 Histopathological pictures of rat pancreas cells after 15 days. (A) Control group. (B) Diabetic control group. (C) Group treated with 2.5 mg kg⁻¹ body weight of CPAM. (D) Group treated with 500 mg kg⁻¹ body weight of metformin hydrochloride. islets of langerhans, acini, vascular congestion. 100×.

Histopathological observations were made on the kidneys of experimental animals after 15 days (Fig. 7A–D). It was observed that for the control specimens, there were large numbers of Bowman's capsules in the section of the cortex of the kidney, and the cells were well rounded throughout the slide (Fig. 7A). However, in diabetic specimens the cells were elongated and had distorted morphology (Fig. 7B). The cell morphology was restored to round in diabetic rats treated with a dose of 2.5 mg kg⁻¹ body weight of CPAM for 15 days (Fig. 7C).

Fig. 7 Histopathological pictures of rat kidney cells after 15 days. (A) Control group. (B) Diabetic control group. (C) Group treated with 2.5 mg kg⁻¹ body weight of CPAM. (D) Group treated with 500 mg kg⁻¹ body weight of metformin hydrochloride. Bowman's capsules. 100×.

3.7 Presence and absorption of CPAM in rat serum samples

The presence of CPAM in the blood serum of treated rats was monitored using MALDI ToF/ToF mass spectrometry and ¹H-NMR spectrometry. The CPAM was detected in blood serum at significant levels (based on mass intensity) up to 6 h after administration, as shown in ESI Fig. 3.†

The signal intensity of the peak at m/z 352.52 ([M + H]⁺) in the MALDI ToF mass spectrum was found to be much higher in the case of serum samples 1 h after treatment (1 × 10⁴ a.u.) than in serum samples 6 h after treatment (340 a.u.), while no such peak was observed in the serum of control rats. It was observed by ¹H-NMR spectroscopy that serum samples from the control group (i.e. untreated rats) had no characteristic peaks of CPAM whereas rats treated with CPAM had characteristic CPAM peaks with δ values from 6.7 to 7.7 ppm (ESI Fig. 4.1 to 4.4†). The peak intensity of CPAM in 1 h serum samples was greater than that in 6 h serum samples (ESI Fig. 4.3 and 4.4†). This implies that the concentration of CPAM was higher in serum 1 h after treatment than 6 h after treatment. The characteristic peaks and δ values from 6.7 to 7.7 ppm matched with our synthetic standard CPAM spectral data, which confirmed that CPAM was present in the blood serum of treated rats (ESI Fig. 4.2 and 4.4†).

3.8 In vitro antibacterial activity of CPAM against Gram-positive, Gram-negative, acid-fast and clinical drug resistant bacteria

The in vitro antibacterial activities of CPAM against a panel of human pathogens belonging to Gram-positive (S. aureus), Gram-negative (P. aeruginosa), acid-fast (M. smegmatis) and multidrug resistant S. aureus (MRSA) were studied by colony forming unit (CFU) assay. CPAM exhibited significant antibacterial activity against the tested microorganisms in a dose-dependent manner. Exposure to 10 μg ml⁻¹ and 50 μg ml⁻¹ of CPAM was found to kill 63% and 67% of the M. smegmatis population after 6 and 24 h respectively (Fig. 8A). Treatment with CPAM at doses of 200 μg ml⁻¹ and 300 μg ml⁻¹ was found to kill 89.1% and 91.5% M. smegmatis population after 6 h, whereas exposure to 100 μg ml⁻¹ and 200 μg ml⁻¹ of CPAM was found to kill 82.9% and 86.5% of the bacterial population after 24 h incubation. In the case of Gram-negative bacteria, 50% of the P. aeruginosa population was killed at doses between 10–50 μg ml⁻¹ and 50–100 μg ml⁻¹ of CPAM after 1 and 3 h respectively (Fig. 8B). Among all tested bacteria, wild type (WT) S. aureus was found to be most susceptible to CPAM with 50% of the bacterial population being killed after treatment with 1 μg ml⁻¹ of CPAM for 6 h, whereas exposure to 100 μg ml⁻¹ and 200 μg ml⁻¹ concentrations killed approximately 95% and 89.2% bacteria after time periods of 6 and 24 h (Fig. 8C). CPAM was also found to be effective against drug resistant S. aureus bacteria such that 50% of S. aureus MRSA was found to be killed at 10 μg ml⁻¹ concentration after 6 h incubation. Treatment with 100 μg ml⁻¹ and 200 μg ml⁻¹ CPAM killed 81.8% and 93.2% bacteria after 6 and 24 h of incubation, respectively (Fig. 8D).

Fig. 8 In vitro activity of CPAM against different bacterial strains at respective time points. (A) M. smegmatis. (B) P. aeruginosa. (C) S. aureus WT. (D) S. aureus MRSA. Bacteria were incubated with different concentrations of CPAM. Values are the means ± S.D. of three independent experiments. Results were found to be statistically significant (p ≤ 0.05 (*), p ≤ 0.001 (**) and p ≤ 0.0001 (***)) compared to those of the control group without treatment with CPAM.

4. Discussion

Compounds having radical scavenging activity could help to reduce unnecessary stress (protein damage, DNA damage or mitochondrial dysfunction) inside cells. The EC₅₀ value of CPAM (10.25 ± 0.89 μM) was less than those of caffeic acid (14.26 ± 1.07 μM) and vitamin E (50.85 ± 1.07 μM) which implies that lower amounts of CPAM are required to neutralize free radicals in comparison to caffeic acid and vitamin E.²¹ Therefore, CPAM is a better antioxidant than caffeic acid and vitamin E. Toxicological analysis of CPAM in Wistar rats suggested that it had no adverse effect on body weight, behaviour and haemoglobin concentration during the experimental treatment period. The four liver function enzymes, namely ACP, ALP, SGPT and SGOT, remained within the normal reference values during the experiment which indicated that CPAM had no adverse effect on rat liver. Similarly, two important kidney function parameters, creatinine and urea, were found to be within the accepted range after treatment, indicating that CPAM had no unfavorable effects on the kidney. These results suggest that CPAM, up to a dosage of 10 mg kg⁻¹ body weight, does not have any toxic effects on the different organs of the rats. The animals remained active and alive even with higher dosages of CPAM throughout the treatment period. From our analysis it is clear that CPAM is a toxicologically safe compound for further study against diabetes. CPAM was effective in significantly reducing blood sugar levels of diabetic rats. Approximately, 90% of the fasting blood sugar levels decreased to normal after treatment with CPAM at a dosage of 2.5 mg for 15 days. The survival of the diabetic animals clearly showed that the dosage of 2.5 mg kg⁻¹ body weight of CPAM was sufficient for the treatment of diabetes. It is well accepted that for diabetic patients, ALP and ACP levels increase.³⁵ To verify this, two different kits were used to measure ALP and ACP values. The experimental results remained normal for toxicological analysis and post diabetes liver function analysis in rats treated with CPAM. The ALP, ACP and prostatic ACP results clearly indicated that CPAM could regulate these parameters. Moreover, CPAM exhibited potent in vitro antibacterial activity against a panel of pathogenic Gram-positive, Gram-negative and acid-fast bacteria. Among all the tested bacteria, CPAM was found to be very effective against S. aureus and M. smegmatis strains and the minimum inhibitory concentration (MIC) was found to be less than 10 μg ml⁻¹ after 6 h treatment. The compound CPAM demonstrated 90% killing of all the tested organisms at concentrations beyond 200 μg ml⁻¹ after certain time periods.

From the results and discussion, we can conclude that CPAM, a catecholic amide, helps to reduce the risk of diabetes in STZ induced rats. Oxidative stress (i.e. free radicals) enhances many complicated diseases along with diabetes in biological systems. Moreover, diabetic patients are more prone to various bacterial infections due to abnormal values of ALP and ACP.³⁶ There is no prior report in the literature that a single molecule CPAM (a catecholic amide) can act as an antioxidant, anti diabetic and antibacterial agent with no toxicological or histopathological adverse effects on the different vital organs of the rat. The small molecular weight of CPAM means that it can be synthesized industrially by a single step using inexpensive reagents. Therefore, the novel CPAM compound can be considered as a lead molecule to control diabetes following further investigations on higher mammalian models.

Ethical statement

Animals were maintained in accordance with the approved guidelines of the Jadavpur University, Kolkata, India and approved by the institutional ethical committee (IEC) of Jadavpur University, Kolkata, India (constituted as per the “Gazette of India” notification part II Sec. 3 (ii) 17 of the Ministry of Environment & Forestry, Government of India, dated 8^th September 1998 for the “The Prevention of Cruelty to Animals Act, 1960”).

Author contributions

K. M. & A. N. conceived the project and designed the research. K. M. performed most of the chemistry experiments and some of the biology experiments and wrote the manuscript. A. S. performed the antibacterial experiments. A. N. and all authors revised the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Acknowledgements

The author Kaushik Misra is indebted to UGC, Delhi, India, for financial support for this research. The authors are highly grateful to Prof. Brajadulal Chattopadhyay and Prof. Abiral Tamang (Department of Bio-Physics, Jadavpur University, Kolkata-700032, West Bengal, India) for helping with in vivo experiments. The authors are thankful to Dr Samiran Sona Gauri (Department of Biotechnology, IIT-Kharagpur 721302, West Bengal, India) for supporting the experiment for detection of CPAM in blood samples.

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Footnote

† Electronic supplementary information (ESI) available. CCDC 1019363 of the crystal of N-(2-bromo-4-fluorophenyl)-3-(3,4-dihydroxyphenyl)-acrylamide (CPAM). For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra16222c

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